Phytochemical analysis and bioactivity screening of three medicinal plants of Saudi Arabia

Purpose: To investigate the phytochemical analysis and bioactivity screening of some Asteraceae medicinal plants. Methods: The chemical constituents were isolated by column chromatography and elucidated using chemical and extensive spectroscopic methodologies including gas chromatoraphy-mass spectrometry (GC-MS), Fourier transform infrared spectroscopy (FTIR), as well as 1D and 2D nuclear magnetic resonance (NMR). The plant extracts were obtained by solvent extraction method while hydrodistillation was used to isolate plant essential oils. Furthermore, cup-plate agar diffusion was applied for antimicrobial activity evaluation while minimum inhibitory concentration (MIC) was assessed by microdilution technique. Results: Centaurea pseudosinaica, Tripleurospermum auriculatum, and Koelpinia linearis afforded previously undescribed three coumarins (xanthotoxin, cirsimaritin, salvigenins) from C. pseudosinaica, one steroid (estradiol) and a pentacyclic triterpene (β-amyrin) from T. auriculatum and a coumarin (santin) from K. linearis in good yields. In addition, the plant extracts and oils exhibited remarkable bioactivities including antifungal, antibacterial and antipyretic etc. Conclusion: The results reveal the presence of bioactive phytomolecules from Asteraceae plant extracts and volatile oils from three Asteraceae plants.


INTRODUCTION
Asteraceae or sunflower is a big and widespread family of flowering plants with more than 1,911 genera and 32,913 species [1]. The useful chemotaxonomic markers of the genus Centuarea are characterized by the presence of terpenoids, flavonoids, alkaloids, coumarins, and sesquiterpene lactones [2]. Centaurea pseudosinaica is native to Middle East and locally abundant mainly in the northern region of Saudi Arabia. To the best of our knowledge, this is the first phytochemical investigation of C. pseudosinaica and its bioactivities except one previous study on the antibacterial activity of this plant against the Gram-+ve bacteria [3].
Nothing appears to have been published before on the chemical constituents of T. auriculatum, although the genus Tripleurospermum has been previously studied and several significant medicinal properties including anti-inflammatory, anti-fungal, anti-bacterial and anti-oxidant were reported [4]. The plant K. linearis, a unique species in the genus Koelpiniais is a rich source of triterpenoids, steroids and series of five longchain alkanoic acid esters of lupeol and lupenone [5]. The purpose of our present study is to determine the phyto-chemical constituents of the plants mentioned above and the evaluation of their bioactivities.

EXPERIMENTAL Plant collection, identification and extract preparation
The entire plant materials including stems, leaves and flowers of C. pseudosinaica, T. auriculatum and K. linearis were procured from Central Saudi Arabia (Riyadh) in early May 2014. Identification of the plant specimen was performed by a taxonomist (Dr Jacob T. Pandalayil) from Herbarium Division, College of Science, KSU, Riyadh, KSA. Samples of the plants were kept in our laboratory under the specimens numbers HZK-121, HZK-122 and HZK 123, respectively. The plants materials were air-dried in shade, crushed into fine powder and then extracted with methanol. The methanolic extracts were filtered, dried and subjected to column chromatography. The hydro-distillation of C. pseudosinaica (whole plant, 90 gm) in a Clevenger-type apparatus afforded yellow colored oil in the yield of 0.67 % v/w (fresh weight basis).

Antimicrobial screening
The hydrodistilled oil and extracts of C. pseudosinaica and others plant extracts T. auriculatum, K. linearis were taken for antimicrobial screening by just dissolving 20 mg (oil/extract) in 1 mL dimethylformamide (DMF) and 50 μL was applied (equivalent to 1mg). The cup-plate agar diffusion technique was used for the antimicrobial screening [24]. The selected pathogens were fungi for example Aspergillus fumigates (RCMB 02568), Syncephalastrum racemosum (RCMB 05922), Geotricum candidum (RCMB 05097), Candida albicans (RCMB 05036) and bacteria such as Gram +ve bacteria (Streptococcus pneumonia (RCMB 010010), Bacillis subtilis (RCMB 010067) and Gram -ve bacteria (Pseudomonas aeruginosa (RCMB 010043), Escherichia coli (RCMB 010052) which were obtained from stock culture at College of Science, KSU, Riyadh. For the positive controls, amphotericin B in DMF (30 μg) for fungi, ampicillin in DMF (30 μg) for Gram +ive bacteria and gentamicin in DMF (30 μg) for Gram -ive bacteria were used as reference antibiotics. The incubation of plates for fungi was done for 72 hrs at 28 o C while one day at 37 o C for bacteria and subsequently inhibition zones were detected and recorded. The minimum inhibitory concentration (MIC) was determined with microdilution method [25a] using serially diluted (2-fold) of plant extract. Mice (200-250g) used for in-vivo experiment were taken from the Veterinary Section (animal house), College of Science, KSU, Riyadh and weighed form 200 to 250g. Before inoculation, they were initially tested for negative pyretic where yeast induced pyrexia (10 ml/kg) was suspended in 0.9 % saline. The determination of bilirubin, albumin, ALT (alanine transaminase), AST (aspartate transaminase), total protein, urea and creatinine was done through standard protocols [25b]. The infective dose (sample) was prepared as an organism loopful (placed in agar slant), transferred into 10 ml test-tube with sterilized peptone water which was then incubated at 36.5 o C for 1 day.

Statistical analysis
The results from the antimicrobial, antipyretic and MIC studies were demonstrated as mean ± and the standard deviation of the triplicate results were calculated and plotted using software Microsoft Excel 2016. The standard deviation was found to be well within the acceptable range as demonstrated in the plot.

Chemical constituents
Bioactive coumarins: xanthotoxin, (a phototoxic furanocoumarin), salvigenin (7-O-methylated flavonoid lipid molecule) and cirsimaritin or skrofulein were isolated from C. pseudosinaica in good yield after recrystallization from alcohol. Another bioactive coumarin, santin was isolated in excellent yield first time from the whole genera Tripleurospermum. Like-wise, a steroid (estradiol -a female sex harmone), and β-amyrin (a pentacyclic triterpene) were obtained from the genus Koelpinia in high yields. Phytochemical screening of the three plants studied in our present study showed the presence of glycosides, terpenes, steroids, tannins, flavonoids (except in K. linearis), coumarins (except in T. auriculatum), chlorides and oxalates while alkaloids and anthraqinones were not present (Table 1). These variations in phytochemical content of the plant are believed due to different environmental factors.

Bioactivity screening
The susceptibility of different pathogens to the inhibitory effect of oils and extracts from C. pseudosinaica and extracts of T. auriculatum, K. linearis have been established and found to show various significant activities for biological applications. As can be seen in Table 2,  significant anti-fungal activities were demonstrated for both hydrodistilled oils and alcoholic extracts of all three plants against vital human pathogens like A. fumigates, S. racemosum, G. candidum and C. albicans. In addition, comparison with the standard antibiotic amphoterocin B (30 µg) clearly revealed that alcoholic extracts showed overall excellent activities against all microorganisms especially against C. albicans where the activities increased significantly. Similar control was observed for anti-bacterial activities for plant species that showed remarkable results when compared with standard broad spectrum antibiotics (ampicillin and gentamycin) against Gram +ve bacteria such as Streptococcus pneumoniae and Bacillis subtilis and Gram -ve bacteria like Pseudomonas aeruginosa and Escherichia coli) whereas, K. linearis exhibited prominent activities as compared to T. auriculatum against all pathogens (fungi and bacteria).
In order to explore the pathogens sensitivity, the in vitro minimum inhibitory concentration (MIC) analyses was examined to evaluate plant oils and extracts effectiveness. As depicted from (Table 3) compared to the controlled reference antibiotics such as amphotericin, ampicillin and gentamycin which showed an encouraging counter effect against all pathogens whereas C. albicans and P. aeruginosa were completely resistant to K. linearis extract. Similarly, Gram +ve and Gram -ve bacteria were also found resistant to C. pseudosinaica oils.

Antipyretic effect of plant extracts
The in-vivo antipyretic effect of plant extracts has revealed that the intake of infective dose of these extracts into mice with yeast induced pyrexia is safe and does not affect the functionality of the kidney and liver. From Table  4, it is shown that the plant extracts' efficiency in lowering the temperature of mice with respect to the standard drugs i.e. paracetamol and aspirin, is fully sensitive and quite effective. The rectal temperature of mice was fluctuating from 1 to 2 o C after regular time intervals but surprisingly it remained constant and control after 16h of treatment as compared to Paracetamol. Similarly, as compared to aspirin, plant extracts have also shown the controlled antipyretic effect when they used the average standard doses of 400 mg/kg.
In another experiment, the in-vivo pharmacologic effect of plant extracts on hepato-renal variation of mice induced with yeast, where concentration of C. pseudosinaica, T. auriculatum and K. linearis extracts have shown a balanced effect as compared to the controlled treatment ( Table 5). The average values for each of ALT, AST, bilirubin, total  protein, albumin, urea and creatinine during 35 days dose at 400 mg/kg were considered for the study. These findings further demonstrated that the functionality of kidney and liver were remained basically unchanged in mice after the intake of these plant extracts.

DISCUSSION
As an ongoing research on the medicinal plants of Saudi Arabia [6][7][8], a first time detailed phytochemical study on some Asteraceae plants growing in Saudi Arabia has been described in this report. The chemical constituents explored in this study and their characterization were compared with the literature.
To the best of our knowledge, C. pseudosinaica has not been previously evaluated for its phytochemical constituents. All three coumarins were isolated in good yield and have been reported to exhibit anti-microbial and antiproliferative activities [9,10]. Likewise, Santin from T. auriculatum, a steroids (estradiol) and a triterpene (β-amyrin) from K. linearis were isolated, characterized and exhibited antioxidant, neuroprotection, anti-ulcer and antimicrobial activities [13,14].
The findings of the results in the current investigation revealed that the variations in bacterial response to the respective plants (oil and extracts) might be due to the structural difference of bacteria and the constituents mode of action against these bacterial species. The notable antimicrobial (antifungal and antibacterial) bio-activity of the studied plants is attributed to the phytomolecules due to unfavourable desert's environment. These results revealed the occurrence of some bioactive volatile/non-volatile compounds in the plant essential oils and extracts could be used to develop anti-microbial agents.

CONCLUSION
In this study, various phytomolecules such as xanthotoxin, cirsimaritin, salvigenins, estradiol, β-amyrin and santin were isolated from three Asteraceae plants of Saudi Arabia. Biological activity evaluation of extracts and essential oils from these plants revealed significant antimicrobial activities and hence, these plants might be used as a suitable candidate for the treatment of microbial diseases.